Project Summary/Abstract The nickel-pincer nucleotide (NPN) cofactor is a newly identified coenzyme discovered in lactate racemase (LarA) from Lactobacillus plantarum. Synthesis of the active enzyme requires the participation of three accessory proteins that act in sequence: LarB carboxylates the pyridinium ring and hydrolyzes the phosphoanhydride of nicotinic acid adenine dinucleotide, LarE converts the two pyridinium ring carboxylates to thiocarboxylates, and LarC inserts nickel (forming two S-Ni and one C-Ni bonds) during synthesis of the novel cofactor. Genes encoding these four proteins are widely distributed in microorganisms associated with the human microbiome and among human pathogens. The long-term objective of the effort described here is to advance significantly our understanding of how microorganisms, including pathogenic species, make and utilize the NPN cofactor. Two specific aims will achieve this objective: (1) characterize the components of the NPN biosynthetic systems and (2) identify the roles of the NPN cofactor in lactate racemase and additional enzymes. Investigations of LarB will define the structure and mechanism of this pyridinium ring carboxylase/phosphoanhydride hydrolase. Studies of a multi-cysteine and probable [4Fe4S]-containing form of LarE will establish whether it operates by a catalytic sulfur-transfer mechanism, in contrast to the sacrificial LarE of L. plantarum with its single active site cysteine that converts to dehydroalanine. Structural and mechanistic analysis of the CTP-dependent nickel-inserting LarC will elucidate how this protein installs nickel into the cofactor. The geometry of lactate binding to L. plantarum LarA will be defined, the full range of substrates used by this enzyme will be established, and substrates will be identified for alternative LarA-like proteins. Proteins that covalently bind the NPN cofactor will be identified and characterized using innovative chemistry that reacts the coenzyme with a fluorescent tag. Radioactive nickel (63Ni) and 14C-nicotinic acid also will be used to label new NPN cofactor-binding proteins, followed by mass spectrometry, bioinformatics, and biochemical studies to identify the functions of these novel enzymes. The findings obtained through these efforts will greatly increase knowledge of the synthesis and utilization of nickel- pincer cofactors in bacteria, including those important to human health, with implications for identification of potential antimicrobial drug targets.